SOMATOSTATIN RECEPTORS IN NEURO2A NEUROBLASTOMA-CELLS - OPERATIONAL CHARACTERISTICS

1 We have used somatostatin (SRIF) receptor subtype-selective ligands to determine some of the operational characteristics of somatostatin r eceptors in Neuro2A mouse neuroblastoma cells. The potent SRIF(1)-rece ptor selective ligand, BIM-23027, was able to displace completely the specific binding of radioiodinated somatostatin, [I-125]-Tyr(11)-SRIF- 14, with a pIC(50) of 10.3, suggesting that Neuro2A cells contain pred ominantly receptors of the SRIF(1) receptor group. The rank order of a ffinities for several somatostatin analogues tested in competition stu dies, together with the high affinity of BIM-23027, indicate that the majority of receptors in Neuro2A cells are of the sst(2) subtype. 2 Th e stable radioligand, [I-125]-BIM-23027, bound with high affinity (K-d = 13 pM, B-max= 0.2 pmol mg(-1) protein) to Neuro2A cell membranes, b ut its binding was only partially reversible at room temperature and b elow. Thus at 4 degrees C, only 36% of the bound ligand dissociated wi thin 2 h. In contrast, 60% of the ligand dissociated at 15 degrees C a nd 89% of the ligand dissociated at 37 degrees C. 3 Equilibrium bindin g of [I-125]-BIM-23027 was partially (25%) inhibited by 10 mu M GTP, a nd by 120 mM NaCl (42% inhibition) but this inhibition was increased t o 75% when sodium chloride and GTP were added together. This effect of GTP and sodium chloride was also seen in dissociation experiments. Af ter incubation to equilibrium with [I-125]-BIM-23027, dissociation was initiated with excess unlabelled ligand in the presence of GTP (10 mu M) and sodium chloride (120 mM). Under these conditions 67% of the li gand dissociated at 4 degrees C, 81% at 15 degrees C and 93% at 37 deg rees C. Binding was totally inhibited by pretreatment of cells with pe rtussis toxin. 4 Functionally, BIM-23027 inhibited forskolin-stimulate d cyclic AMP accumulation in a concentration-dependent manner with an IC50 of 1.0 nM and a maximal inhibition of 37%. This effect was abolis hed by pretreatment of the cells with pertussis toxin. However, unlike in studies reported with the recombinant sst(2) receptor, no rise in intracellular calcium concentration was observed with SRIF-14. 5 We co nclude that Neuro2A cells provide a stable neuronal cell line for the study of functionally coupled endogenous somatostatin receptors of the sst(2) type. In addition, we have found that activation of the recept or is associated with ligand-receptor internalisation.